Method for detecting target rna by utilizing nicking/extension chain reaction system-based isothermal nucleic acid amplification

ABSTRACT

The present invention relates to a detection method for detecting a target RNA contained in a sample with high sensitivity by using nicking/extension chain reaction system-based isothermal nucleic acid amplification (NESBA) that uses activity of a cleavage enzyme and a DNA polymerase. The NESBA of the present invention is a new concept isothermal target RNA detection method that realizes higher amplification efficiency than the existing NASBA technology and is deemed to be utilizable as a new concept diagnosis technology that can replace conventional target RNA detection technologies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional under 35 USC §120 of U.S. patent application Ser.No. 16/958,683 filed Jun. 27, 2020, which in turn is a U.S. nationalphase under the provisions of 35 U.S.C. §371 of International PatentApplication No. PCT/KR18/16946 filed Dec. 31, 2018, which in turn claimspriority under 35 U.S.C. §119 of Korean Patent Application No.10-2017-0183675 filed Dec. 29, 2017. The disclosures of all suchapplications are hereby incorporated herein by reference in theirrespective entireties, for all purposes.

REFERENCE TO SEQUENCE LISTING

This application includes an electronically submitted sequence listingin .XML format. The .XML file contains a sequence listing entitled“521DIV_SeqListing.xml” created on Sep. 6, 2022 and is 4096 bytes insize. The sequence listing contained in this .XML file is part of thespecification and is hereby incorporated by reference herein in itsentirety.

TECHNICAL FIELD

The present invention relates to a method of amplifying and detecting atarget RNA, and more particularly to a method of amplifying anddetecting a target RNA usingnicking/extension-chain-reaction-system-based isothermal nucleic acidamplification (NESBA).

BACKGROUND ART

As living quality has improved, modern people's interest in earlydiagnosis of disease has grown, and such interest has a direct influenceon the growth of the diagnosis market. According to the Frost & Sullivansurvey, the global in-vitro diagnosis market recorded an annual growthrate (CAGR) of 7.3%, starting at about 47 billion dollars in 2013 andreaching a scale of about 63 billion dollars in 2017. In addition, amongvarious technical fields in the rapidly growing in-vitro diagnosismarket, molecular diagnostic and on-site diagnostic fields have beenobserved to be the technical fields recording the highest growth rates,namely 12.7% and 8.4%, respectively. Molecular diagnostic technologyenables the direct detection of genetic information (DNA/RNA) of apathogen that causes a disease, and thus is receiving much attention asa technology capable of addressing disadvantages of immunodiagnostictechnology for detecting indirect factors of a disease on the basis ofknown antigen/antibody reactions. In addition, on-site diagnostictechnology is a technology that can remarkably reduce the time andexpense associated with use of large hospitals or specialized diagnosticcenters for checkups, unlike conventional diagnostic technologies.

With the rapid advance in these molecular diagnostic and on-sitediagnostic technical fields, the severity of disease-related problemscaused by RNA viruses has recently emerged. An example that can show theseverity of RNA virus diseases is the 2015 Middle East RespiratorySyndrome (MERS). MERS is a viral respiratory infection caused bycoronavirus (a type of RNA virus), and in 2015, 1,599 cases of MERSinfections occurred worldwide, and a fatality rate of about 40% wasrecorded (as of Jun. 26, 2015). In the same year in Korea, 186 MERScases occurred as of July 5, starting with the first confirmed patienton May 20, and 38 deaths occurred by November. As such, in the case ofdiseases caused by RNA viruses, great human damage easily occurs, andthis can be said to result from a high mutation rate, which is acharacteristic of RNA viruses. Due to this characteristic of RNAviruses, it is difficult to diagnose whether or not an infection occurs,and as a result, it is difficult to diagnose and deal with a diseaseearly.

Technology has been developed to address these problems, and amongvarious technologies that have been developed to date, the mostrepresentative diagnostic technology used in RNA virus detection is areverse transcription polymerase chain reaction (RT-PCR). RT-PCR is atechnique of generating complementary DNA (cDNA) from target RNA using areverse transcriptase before a DNA amplification reaction and thenamplifying the produced complementary DNA through PCR. This technique isadvantageous in that a target DNA can be detected with high sensitivityusing the high amplification efficiency of PCR, and is a technique thathas been essentially used to confirm a disease to date. However, inorder to implement such a RT-PCR technique, sophisticated temperaturecontrol for a nucleic acid amplification reaction is required, whichacts as a factor that hinders the miniaturization of diagnostic devices.Therefore, the RT-PCR technique is disadvantageous in that it can beused only in limited facilities such as large hospitals or specializeddiagnostic centers equipped with diagnostic equipment.

As an RNA detection technique capable of addressing the disadvantage ofthe aforementioned RT-PCR technique, nucleic-acid-sequence-basedamplification (hereinafter referred to as NASBA) was developed (Kievitset al., Journal of Virological Methods, 35:273, 1991). NASBA involvesamplification and detection reactions of target RNA under isothermalconditions (41° C.), and thus has been used as a new isothermal nucleicacid amplification technique capable of addressing the problems ofexisting RT-PCR. NASBA is based on a reaction in which aT7-promoter-containing double-stranded DNA is produced from target RNA,and then a large amount of anti-sense RNA is produced through a T7 RNApolymerase transcription reaction. In addition, the produced anti-senseRNA can be used as a substrate for the production reaction of aT7-promoter-containing double-stranded DNA, thereby implementing anamplification reaction with high efficiency. However, NASBA isdisadvantageous in that the anti-sense RNA amplification reaction isdependent only on the transcription reaction of T7 RNA polymerase. Thus,when the efficiency of the reaction in which a T7-promoter-containingdouble-stranded DNA is produced from target RNA is low, a problem mayalso occur in the efficiency of the anti-sense RNA amplificationreaction based on the transcription reaction of T7 RNA polymerase, whichmay act as a factor that generates a false signal.

Therefore, as a result of having made intensive efforts to address theabove-described problems with the related art, the inventors of thepresent invention confirmed that, by introducing a primer modified withan nicking enzyme recognition nucleotide sequence into existing NASBA,the exponential amplification reaction of a T7-promoter-containingdouble-stranded DNA was performed on the basis of nicking enzyme and DNApolymerase activity, and then an anti-sense RNA was produced, and as aresult, a new concept isothermal nucleic acid amplification reactionwith high amplification efficiency compared to the existing NASBA can beimplemented, and thus completed the present invention.

DISCLOSURE Technical Problem

Therefore, the present invention has been made in view of the aboveproblems, and it is an object of the present invention to provide amethod of increasing target RNA detection sensitivity by enhancing thenucleic acid amplification reaction efficiency of existing NASBA throughnicking/extension-chain-reaction-system-based isothermal nucleic acidamplification.

Technical Solution

In accordance with an aspect of the present invention, the above andother objects can be accomplished by the provision of a method ofdetecting of a target RNA based on a nicking/extension chain reactionsystem, comprising:

(a) hybridizing a target RNA with a first primer including a sequencecomplementary to the target RNA, a T7 promoter sequence, and a DNAnicking enzyme recognition nucleotide sequence, and then producingcomplementary DNA of the target RNA using a reverse transcriptase;

(b) degrading the target RNA bound to the complementary DNA produced instep (a) by a ribonuclease;

(c) hybridizing the complementary DNA with a second primer including asequence complementary to the complementary DNA and a DNA nicking enzymerecognition nucleotide sequence, and then producing a double-strandedDNA including a T7 promoter and the nicking enzyme recognitionnucleotide sequence using a reverse transcriptase;

(d) treating the double-stranded DNA produced in step (c) with a DNAendonuclease to cleave a DNA nicking enzyme recognition site of thedouble-stranded DNA, and then amplifying the T7-promoter-containingdouble-stranded DNA using DNA polymerase; and (e) producing ananti-sense RNA from the T7-promoter-containing double-stranded DNAamplified in step (d) using T7 RNA polymerase, and then detecting theproduced anti-sense RNA.

In accordance with another aspect of the present invention, there isprovided a composition for detecting a target RNA, comprising: (i) afirst primer comprising a sequence complementary to the target RNA, a T7promoter sequence, and a DNA nicking enzyme recognition nucleotidesequence; and (ii) a second primer comprising a sequence complementaryto DNA complementary to the target nucleic acid and a DNA nicking enzymerecognition nucleotide sequence.

In accordance with a further aspect of the present invention, there isprovided a kit for detecting a target RNA, comprising: (i) a firstprimer comprising a sequence complementary to the target RNA, a T7promoter sequence, and a DNA nicking enzyme recognition nucleotidesequence; and (ii) a second primer including a sequence complementary toDNA complementary to the target nucleic acid and a DNA nicking enzymerecognition nucleotide sequence.

DESCRIPTION OF DRAWINGS

The above and other objects, features and other advantages of thepresent invention will be more clearly understood from the followingdetailed description taken in conjunction with the accompanyingdrawings, in which:

FIG. 1 illustrates a NESBA reaction of the present invention, wherein(A) illustrates a reaction in which a T7-promoter-containingdouble-stranded DNA is produced from a target RNA, (B) illustrates areaction in which the T7-promoter-containing double-stranded DNAproduced as illustrated in (A) is exponentially amplified through anicking/polymerase chain reaction, and (C) illustrates a reaction inwhich an anti-sense RNA is produced from the T7-promoter-containingdouble-stranded DNA produced as illustrated in (B) through the activityof T7 RNA polymerase;

FIG. 2 illustrates the results of comparing the amplification efficiencyof NESBA of the present invention with that of existing NASBA;

FIG. 3 illustrates the results of confirming target RNA detectionsensitivity of NESBA of the present invention; and

FIG. 4 illustrates the results of confirming target RNA detectionsensitivity using reverse transcription PCR.

DETAILED DESCRIPTION AND EXEMPLARY EMBODIMENTS

Reference will now be made in detail to the preferred embodiments of thepresent invention, examples of which are illustrated in the accompanyingdrawings. Wherever possible, the same reference numbers will be usedthroughout the drawings to refer to the same or like parts.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which the present invention pertains. In general, thenomenclature used herein and experimental methods described below arewell known and commonly used in the art.

In the present invention, to develop a method of amplifying a final RNAproduct (hereinafter referred to as an anti-sense RNA) produced when atarget RNA is present through T7 RNA polymerase activity at isothermaltemperature and of detecting the amplified anti-sense RNA, without usingexpensive analysis equipment for precisely performing temperaturechanges, by introducing nicking/extension-chain-reaction-system-basedisothermal nucleic acid amplification, a T7-promoter-containingdouble-stranded DNA, which is produced in an existing NASBA technique,was exponentially amplified, and therefore a method of enhancing theproduction efficiency of an anti-sense RNA was developed.

Therefore, the present invention relates to a method of detecting atarget RNA on the basis of a nicking/extension chain reaction system,the method comprising: (a) hybridizing a target RNA with a first primercomprising a sequence complementary to the target RNA, a T7 promotersequence, and a DNA nicking enzyme recognition nucleotide sequence, andthen producing complementary DNA of the target RNA using a reversetranscriptase; (b) degrading the target RNA bound to the complementaryDNA produced in step (a) by a ribonuclease; (c) hybridizing thecomplementary DNA with a second primer comprising a sequencecomplementary to the complementary DNA and a DNA nicking enzymerecognition nucleotide sequence, and then producing a double-strandedDNA comprising a T7 promoter and the nicking enzyme recognitionnucleotide sequence using a reverse transcriptase; (d) treating thedouble-stranded DNA produced in step (c) with a DNA nicking enzyme tocleave a DNA nicking enzyme recognition site of the double-stranded DNA,and then amplifying the T7-promoter-containing double-stranded DNA usingDNA polymerase; and (e) producing an anti-sense RNA from theT7-promoter-containing double-stranded DNA amplified in step (d) usingT7 RNA polymerase, and then detecting the produced anti-sense RNA. (seeFIG. 1 ).

In step (a) of the present invention, the target RNA binds to and ishybridized with a sequence complementary to the target RNA of the firstprimer (sequence complementary to target RNA+T7 promoter sequence+DNAnicking enzyme recognition site sequence), a reverse transcriptase bindsto the first primer that is hybridized with the target RNA to produceDNA complementary to the target RNA, and the target RNA and DNAcomplementary thereto complementarily bind to each other.

In step (b) of the present invention, the target RNA bound to thecomplementary DNA produced in step (a) is degraded with a ribonuclease,and consequently, a DNA strand, including the sequence complementary tothe target RNA, a T7 promoter, and the nicking enzyme recognitionnucleotide sequence, remains.

In step (c) of the present invention, the DNA strand including thesequence complementary to the target RNA, a T7 promoter, and the nickingenzyme recognition nucleotide sequence, which remains in step (b), bindsto and is hybridized with a second primer (sequence complementary to theDNA strand+nicking enzyme recognition sequence), a reverse transcriptasebinds to the hybridized primer to synthesize a complementary DNA strand,and consequently, a double-stranded DNA including the sequencecomplementary to the target RNA, the T7 promoter sequence, and thenicking enzyme recognition sequence is produced.

In step (d) of the present invention, the double-stranded DNA producedin step (c) (a sequence complementary to the target RNA+a T7 promotersequence+an nicking enzyme recognition sequence) is treated with a DNAnicking enzyme that recognizes and cleaves the nicking enzymerecognition sequence, thereby cleaving a single strand of thedouble-stranded DNA. The cleaved DNA strand is used as a primer for newDNA synthesis to synthesize a new DNA strand using DNA polymerasepresent in a sample, and the previously bound DNA strand is released bythe strand displacement activity of the DNA polymerase. The released DNAstrand binds to and is hybridized with a first primer or a second primerthat has not participated in the reaction, is synthesized by DNApolymerase into a double-stranded DNA, and is cleaved by a DNA nickingenzyme, and the DNA polymerase binds to the cleaved strand, therebysynthesizing a new DNA strand, and the previously bound DNA strand isreleased, and these processes are repeated, through which adouble-stranded DNA including a sequence complementary to the target RNAand a T7 promoter is exponentially amplified.

In step (e) of the present invention, T7 RNA polymerase is used toproduce an anti-sense RNA from the double-stranded DNA including thesequence complementary to the target RNA and a T7 promoter, which hasbeen amplified in step (d), and the produced anti-sense RNA is detected.

In the present invention, steps (a) to (e) may be performed atisothermal temperature, preferably at a temperature ranging from 40° C.to 42° C.

In the present invention, the “first primer” has a sequence including asequence complementary to a target RNA, a T7 promoter sequence, and aDNA nicking enzyme recognition nucleotide sequence, and in step (a), thesequence complementary to the target RNA of the first primer and thetarget RNA bind to and are hybridized with each other, and a reversetranscriptase binds to the first primer hybridized with the target RNA,and consequently, DNA complementary to the target RNA is produced fromthe target RNA. In addition, in step (d), the DNA strand released by theaction of the DNA polymerase binds to and is hybridized with a firstprimer or a second primer that has not participated in the reaction, andis synthesized by DNA polymerase into a double-stranded DNA.

In one embodiment of the present invention, for example, a primer of SEQID NO: 1 below is used as the first primer, wherein the italic portion(GG ATC) denotes a DNA nicking enzyme recognition site, the underlinedbold portion denotes a T7 promoter site (TAA TAC GAC TCA CTA TAG), andthe “GGG ATG CTT GAG CAT ACA GG” site is a site complementary to thetarget RNA.

First primer:  (SEQ ID NO: 1) 5′-AAA AAA A

 GGG GAA TTC  TAA TAC  GAC TCA CTA TAG  GGG ATG CTT GAG CAT AGA GG 

In the present invention, the above sequence is used as an example, butthe sequence of used primer varies depending on the types of target RNAand DNA nicking enzyme.

In the present invention, the “second primer” has a sequence including asequence complementary to DNA complementary to the target RNA and annicking enzyme recognition nucleotide sequence, and in step (c), thesecond primer binds to and is hybridized with the remaining “DNA strandincluding the sequence complementary to the target RNA, a T7 promoter,and the nicking enzyme recognition nucleotide sequence,” a reversetranscriptase binds to the hybridized primer to synthesize acomplementary DNA strand, and consequently, a double-stranded DNAincluding the sequence complementary to the target RNA, a T7 promotersequence, and the nicking enzyme recognition sequence is produced. Inaddition, in step (d), the DNA strand released by the action of the DNApolymerase binds to and is hybridized with a first primer or a secondprimer that has not participated in the reaction, and is synthesized bythe DNA polymerase into a double-stranded DNA.

In one embodiment of the present invention, for example, a primer of SEQID NO: 2 below is used as the second primer, wherein the italic portion(GG ATC) denotes a DNA nicking enzyme recognition sequence, and theunderlined portion (GGG GAG CTC TGC TTG CAT AAG G) denotes a sequencecomplementary to DNA complementary to the target RNA.

Second primer:  (SEQ ID NO: 2) 5′-AAA AAA A

 GGG GAG CTC TGC TTG CAT AAG G-3′

In the present invention, the above sequence is used as an example, butthe sequence of used primer varies depending on the types of target RNAand DNA endonuclease.

In the present invention, the ribonuclease is not limited, as long as itis an enzyme that selectively degrades only RNA in a DNA/RNA hybrid, andmay be RNaseH, RNaseT1, or the like, but preferably, RNaseH or the likemay be used.

In the present invention, the DNA polymerase is not limited, as long asit is a DNA polymerase having strand displacement activity on DNA boundto a template, and preferably, a Klenow fragment or the like may beused.

Examples of various DNA polymerases that may be used in the presentinvention include a Klenow fragment of E. coli DNA polymerase I, athermostable DNA polymerase, and a bacteriophage T7 DNA polymerase.Preferably, the polymerase is a thermostable DNA polymerase that can beobtained from various bacteria species, and examples thereof includeThermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis,Thermus flavus, Thermococcus litoralis, and Pyrococcus furiosus (Pfu).When a polymerization reaction is performed, components required for thereaction may be provided in excess amounts in a reaction container.Excess amounts of components required for an amplification reactionrefer to amounts such that the amplification reaction is notsubstantially limited by the concentrations of the components.

In the present invention, the DNA nicking enzyme may be a nickingendonuclease, and is not limited, as long as it is an enzyme thatrecognizes and cleaves the specific recognition site of a DNA nucleotidesequence. The nicking endonuclease is an enzyme that catalyzesrecognition of the specific nucleotide sequence of a double-stranded DNAmolecule and cleavage of a portion thereof or the periphery thereof, andmost nicking endonucleases cleave DNA at a location with a specificnucleotide sequence, which is referred to as a recognition site or arestriction site.

The site recognized by a nicking endonuclease has a specific sequencecalled a palindrome. That is, this means that the recognition site hasthe same nucleotide sequence in the 5′ to 3′ direction of both strands.The specific nucleotide sequence denotes a recognition site. Forexample, the enzyme EcoRI cleaves DNA only when it meets the nucleotidesequence of GAATTC in a DNA double helix to form a 5′-overhanging stickyend. In the present invention, the nicking endonuclease is not limited,as long as it is an enzyme that recognizes and cleaves the specificrecognition site of a DNA nucleotide sequence, and may be, for example,EcoRI, BamHI, HindiIII, KpnI, NotI, PstI, SmaI, XhoI, or the like.

In the present invention, “anti-sense” refers to an oligomer with asequence of nucleotide bases and a backbone between sub-units that allowthe anti-sense oligomer to hybridize with a target sequence in RNA byWatson-Crick base pairing, to form a RNA:oligomer heteroduplex withinthe target sequence, typically with mRNA. The oligomer may have exactsequence complementarity to the target sequence or near complementarity.It is required to add co-factors such as Mg²⁺, dATP, dCTP, dGTP, anddTTP to a reaction mixture to the extent to which a desiredamplification degree can be achieved. All enzymes used in theamplification reaction may be active under the same reaction conditions.In fact, buffers provide conditions close to an optimum reactionconditions for all enzymes. Thus, the amplification process of thepresent invention may be carried out on a single reactant withoutchanging conditions such as adding reactants.

According to one embodiment of the present invention, a molecular beaconincluding a sequence complementary to the target RNA is used as a meansfor detecting an anti-sense RNA. In addition, various types of probesthat bind to the target nucleic acid may be used, and the term “probe”as used herein refers to a single-strand nucleic acid molecule whichincludes a sequence complementary to a target nucleic acid sequence.According to one embodiment of the present invention, the probes of thepresent invention may be modified within the scope that does not impairthe advantages of the probes of the present invention, i.e., improvementin hybridization specificity. These modifications, i.e., labels, mayprovide a signal to detect the presence or absence of hybridization, andmay be linked to oligonucleotides. Suitable labels include fluorophores(e.g., fluorescein, phycoerythrin, rhodamine, lissamine, and Cy3 and Cy5(Pharmacia)), chromophores, chemi-luminophores, magnetic particles,radioisotopes (P³² and S³⁵), mass labeling, electron dense particles,enzymes (alkaline phosphatase or horseradish peroxidase), cofactors,substrates for enzymes, heavy metals (e.g., gold) and hapten, withspecific binding partners such as antibodies, streptavidin, biotin,digoxigenin and chelating groups, but the present invention is notlimited thereto. Labeling may be performed using various methodscommonly used in the art, for example, a nick translation method, arandom priming method (Multiprime DNA labelling systems booklet,“Amersham” (1989)), and a Kination method (Maxam & Gilbert, Methods inEnzymology, 65:499(1986)). Labels provide signals detectable usingfluorescence, radioactivity, colorimetry, gravimetric measurement, X-raydiffraction or absorption, magnetism, enzymatic activity, mass analysis,binding affinity, hybridization high frequency, and nanocrystals.

According to one embodiment of the present invention, a probe may beused as a means for detecting an anti-sense RNA, and the probe of thepresent invention may be immobilized on a water-insoluble carrier (e.g.,a nitrocellulose or nylon filter, a glass plate, a silicone and afluorocarbon support) to be manufactured as a microarray. In themicroarray, the probe of the present invention is used as a hybridizablearray element. The immobilization onto the water-insoluble carrier isperformed by a chemical bonding method or a covalent bonding method suchas UV. For example, the hybridizable array element may bind to a glasssurface modified so as to include an epoxy compound or an aldehydegroup, and may also bind to a polylysine coating surface by UV. Inaddition, the hybridizable array element may bind to a carrier via alinker (e.g., an ethylene glycol oligomer and a diamine). When the probeis used, the probe is hybridized with an anti-sense RNA molecule. In thepresent invention, suitable hybridization conditions may be determinedthrough a series of processes by an optimized procedure. For example,conditions such as temperature, the concentration of components,hybridization and washing time, buffer components, and pH and ionicstrength thereof depend on various factors such as the length of aprobe, GC content, a target nucleotide sequence, and the like. Thedetailed conditions for hybridization can be confirmed in JosephSambrook, et al., Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); and M. L. M.Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc. N.Y.(1999). After the hybridization reaction, a hybridization signalgenerated through the hybridization reaction is detected. Detection ofhybridization signaling may be performed using various methods, forexample, depending on the type of label bound to the probe. For example,when the probe is labeled with an enzyme, a substrate of the enzyme maybe reacted with a hybridization reaction product to confirm the presenceor absence of hybridization. Suitable combinations of enzyme/substrateinclude a peroxidase (e.g., horseradish peroxidase) and chloronaphthol,aminoethylcarbazole, diaminobenzidine, D-luciferin,bis-N-methylacridinium nitrate (lucigenin), resorufin benzyl ether,luminol, an Amplex red reagent (10-acetyl-3,7-dihydroxyphenoxazine),p-phenylenediamine-HCl and pyrocatechol (HYR), tetramethylbenzidine(TMB), 2,2′-azine-di[3-ethylbenzthiazoline sulfonate] (ABTS),o-phenylene diamine (OPD), and naphthol/pyronine; alkaline phosphataseand bromochloroindolyl phosphate (BCIP), nitroblue tetrazolium (NBT),naphthol-AS-B1-phosphate and ECF substrate; glucose oxidase andnitroblue tetrazolium (t-NBT); and the like. When the probe is labeledwith gold particles, this may be detected by a silver staining methodusing silver nitrate. By analyzing the intensity of the hybridizablesignal through the above-described hybridization process, the target RNAmay be detected.

In the NESBA of the present invention, unlike the existing NASBA,primers including DNA nicking enzyme recognition nucleotide sequencesare used to produce a double-stranded DNA including a T7 promoter andthe DNA nicking enzyme recognition nucleotide sequence. A single strandof the double-stranded DNA is cleaved by the activity of a nickingenzyme recognizing the nicking enzyme recognition nucleotide sequencepresent in a sample. A short DNA strand produced by the cleavagereaction is used as a primer for new DNA synthesis, and thus a new DNAsynthesis reaction proceeds based on the activity of DNA polymerasepresent in the sample. The DNA polymerase used in this reaction hasstrand displacement activity on DNA bound to a template in the DNAsynthesis reaction. Thus, when new DNA is synthesized using the producedshort DNA strand as a primer, the DNA bound to a template is released,and this DNA released from the template may be used as a template towhich a primer that has not participated in the reaction can bind.Accordingly, the nicking/extension chain reaction is implemented throughthe nicking enzyme and DNA polymerase activity, thereby exponentiallyamplifying a T7-promoter-containing double-stranded DNA, which may be acore factor that can realize enhanced amplification efficiency comparedwith existing NASBA reactions.

FIG. 1 illustrates a NESBA technique of the present invention, wherein(A) illustrates a reaction in which a T7-promoter-containingdouble-stranded DNA is produced from a target RNA, (B) illustrates areaction in which the T7-promoter-containing double-stranded DNAproduced as illustrated in (A) is exponentially amplified through anicking/polymerase chain reaction, and (C) illustrates a reaction inwhich an anti-sense RNA is produced from the T7-promoter-containingdouble-stranded DNA produced as illustrated in (B) through the activityof T7 RNA polymerase.

In one embodiment of the present invention, as a result ofexperimentally examining whether or not amplification efficiency of theNESBA technique of the present invention is enhanced, it was confirmedthat the present invention showed a much higher amplification efficiencyin an experiment for detecting a target RNA at the same concentration(H3N2 influenza gRNA, 800 fM) compared to existing NASBA reactions (seeFIG. 2 ).

In addition, in another embodiment of the present invention, it wasconfirmed that the limit of detection (LOD) of target RNA for the NESBAtechnique of the present invention was 0.72 fM, from which it wasconfirmed that the NESBA technique has very high detection sensitivity.These results mean that the NESBA technique of the present invention hasdetection sensitivity similar to that of an existing reversetranscription PCR technique, under isothermal conditions withoutexpensive analysis devices.

Another embodiment of the present invention relates to a composition fordetecting a target RNA, comprising: (i) a first primer comprising asequence complementary to the target RNA, a T7 promoter sequence, and aDNA nicking enzyme recognition nucleotide sequence; and (ii) a secondprimer comprising a sequence complementary to DNA complementary to thetarget nucleic acid and a DNA nicking enzyme recognition nucleotidesequence.

In the present invention, the composition for detecting a target RNA mayfurther comprise a reverse transcriptase, T7 RNA polymerase, aDNAnicking enzyme, DNA polymerase, and dNTP.

Another embodiment of the present invention relates to a kit fordetecting a target RNA, comprising: (i) a first primer comprising asequence complementary to the target RNA, a T7 promoter sequence, and aDNA nicking enzyme recognition nucleotide sequence; and (ii) a secondprimer comprising a sequence complementary to DNA complementary to thetarget nucleic acid and a DNA nicking enzyme recognition nucleotidesequence.

In the present invention, the kit for detecting a target RNA may furthercomprising a reverse transcriptase, T7 RNA polymerase, a DNA nickingenzyme, DNA polymerase, and dNTP.

Hereinafter, the present invention will be described in further detailwith reference to the following examples. It will be obvious to those ofordinary skill in the art that these examples are provided forillustrative purposes only and should not be construed as limiting thescope of the present invention.

EXAMPLE 1 Establishment of Nicking/Extension-Chain-Reaction-System-basedIsothermal Nucleic Acid Amplification Reaction Conditions using NickingEnzyme and DNA Polymerase Activity

A NESBA reaction solution used in the present example was prepared byadding, to a reaction buffer solution, 2.8 μL of rNTPs (25 mM each), 1.4μL of dNTPs (10 mM each), 0.5 μL of a first primer (10 μM), 0.5 μL of asecond primer (10 μM), 1 μL of a molecular beacon (6 μM), and 0.4 μL ofa target RNA (H3N2 influenza virus gRNA), and the reaction buffersolution includes 40.4 mM Tris-HCl (pH 8.2), 50 mM NaCl, 53.2 mM KCl,19.1 mM MgCl₂, 7.7 mM DTT, 11.4% DMSO, and 90 μg/mL BSA. The reactionsolution was heated at 65° C. for 5 minutes, and then slowly cooled to41° C. at a rate of 0.1° C./s and allowed to stand for 5 minutes.Subsequently, 5 μL of a NASBA enzyme cocktail (AMV-RT, RNase H, T7 RNApolymerase), 1.6 μL of a Klenow fragment (3′5′ exo-) (5 unit/μL), and0.5 μL of Nt.AlwI (10 unit/μL) were added to the reaction solution, andthen a reaction was allowed to occur at 41° C. for 2 hours. After thereaction was completed, a fluorescence signal generated from afluorescein (FAM) modified in the molecular beacon was measured toanalyze the amount of anti-sense RNA that was produced.

The nucleotide sequence information used in the present example is asfollows, but the present invention is not limited thereto.

-First primer:  (SEQ ID NO: 1) 5′-AAA AAA AGG ATC GGG GAA TTC TAA TACGAC TCA CTA TAG GGG ATG CTT GAG CAT ACA GG-3′ (SEQ ID NO: 2)-Second primer:  5′-AAA AAA AGG ATC GGG GAG CTC TGC TTG  CAT AAG G-3′-Molecular beacon:  (SEQ ID NO: 3) 5′-[FAM] CCA GCA TTG AAC GTG ACTATG CTG G [DABCYL]-3′

In addition, an anti-sense RNA was produced from the target RNA using aconventional technique, NASBA, as a control. More specifically, a NASBAreaction solution was prepared by adding, to a reaction buffer solution,2.8 μL of rNTPs (25 mM each), 1.4 μL of dNTPs (10 mM each), 0.5 μL of afirst primer (10 μM), 0.5 μL of a second primer (10 μM), 1 μL of amolecular beacon (6 μM), and 0.4 μL of a target RNA (H3N2 influenzavirus gRNA), and the reaction buffer solution includes 40 mM Tris-HCl(pH 8.5), 12 mM MgCl₂, 70 mM KCl, 10 mM DTT, and 15% DMSO. In addition,the first and second primers used in the present NASBA reaction do notinclude an nicking enzyme recognition nucleotide sequence. The reactionsolution was heated at 65° C. for 5 minutes and then slowly cooled to41° C. at a rate of 0.1° C./s, and allowed to stand for 5 minutes.Subsequently, 5 μL of a NASBA enzyme cocktail (AMV-RT, RNase H, T7 RNApolymerase) was added to the reaction solution, and then a reaction wasallowed to occur at 41° C. for 2 hours. After the reaction wascompleted, a fluorescence signal generated from a fluorescein (FAM)modified in the molecular beacon was measured to analyze the amount ofproduced anti-sense RNA.

As a result, as illustrated in FIG. 2 , it was confirmed that the NESBAmethod of the present invention exhibited remarkably high anti-sense RNAamplification efficiency in an experiment for detecting target RNA (H3N2influenza virus gRNA, 800fM) at the same concentration compared with anexisting NASBA reaction.

EXAMPLE 2 Verification of Sensitivity ofNicking/Extension-Chain-Reaction-System-based Isothermal Nucleic AcidAmplification Technique using Nicking Enzyme and DNA Polymerase Activity

Analysis samples (20 μL) containing a target RNA at variousconcentrations (0.1 pM, 0.2 pM, 0.5 pM, 0.8 pM, 8 pM, and 80 μM) wereprepared in the same manner as in Example 1, and then a NESBA reactionwas performed to analyze sensitivity to the target RNA. Morespecifically, after the NESBA reaction, an experiment was performedusing an Infinite M200 PRO (Tecan) to measure a fluorescence signalgenerated from a fluorescein modified in the molecular beacon. Inaddition, a device for measuring the fluorescence signal generated fromfluorescein was set as follows, but the present invention is not limitedthereto.

Excitation wavelength: 480 nm Emission wavelength: 520-600 nm

As a result, as illustrated in FIG. 3 , it was confirmed that the limitof detection (LOD) of the target RNA of the NESBA technique was 0.72 fM.

EXAMPLE 3 Verification of Practicability ofNicking/Extension-Chain-Reaction-System-based Isothermal Nucleic AcidAmplification Technique using Nicking Enzyme and DNA Polymerase Activity

To verify the practicability of the NESBA technique of the presentinvention, an experiment for detecting a target RNA using acommercialized reverse transcription PCR kit was carried out, and theresults were compared with the performance of a NESBA technique. In thepracticability verification experiment of the present example, aTOPrealTM One-step RT qPCR kit (Enzynomics co Ltd.) was used, and thetarget RNA detection experiment was performed according to anexperimental method provided by the above product. As a result ofperforming an experiment for detecting a target RNA at variousconcentrations (0.2 pM, 2 pM, 20 pM, 200 pM, and 2000 pM) by using thereverse transcription PCR kit, it was confirmed that the reversetranscription PCR technique exhibited a limit of detection of the targetRNA of 0.52 fM (see FIG. 4 ), and from these results, it was confirmedthat the NESBA technique of the present invention, which does not useexpensive reverse transcription equipment, exhibited detectionperformance comparable to that of an existing reverse transcription PCRtechnique.

INDUSTRIAL APPLICABILITY

A target RNA detection method using NESBA according to the presentinvention does not require a temperature control reaction, as isrequired in an existing reverse transcription PCR technique, and thuscan be used as a technique for on-site diagnosis, not only as atechnique used in specialized diagnostic centers and large hospitals,can realize enhanced amplification efficiency compared to an existingNASBA reaction, and can address false signal problems that may occur ina target RNA detection process.

While specific embodiments of the present invention have been describedin detail, it will be obvious to those of ordinary skill in the art thatthese detailed descriptions are merely exemplary embodiments and are notintended to limit the scope of the present invention. Therefore, thesubstantial scope of the present invention should be defined by theappended claims and equivalents thereto.

What is claimed is:
 1. A composition for detecting a target RNA, thecomposition comprising: (i) a first primer comprising a sequencecomplementary to the target RNA, a T7 promoter sequence, and a DNAnicking enzyme recognition nucleotide sequence; and (ii) a second primercomprising a sequence complementary to DNA complementary to the targetnucleic acid and a DNA nicking enzyme recognition nucleotide sequence.2. The composition according to claim 1, further comprising a reversetranscriptase, T7 RNA polymerase, a DNA endonuclease, DNA polymerase,and dNTP.
 3. A kit for detecting a target RNA, the kit comprising: (i) afirst primer comprising a sequence complementary to the target RNA, a T7promoter sequence, and a DNA nicking enzyme recognition nucleotidesequence; and (ii) a second primer comprising a sequence complementaryto DNA complementary to the target nucleic acid and a DNA nicking enzymerecognition nucleotide sequence.
 4. The kit according to claim 3,further comprising a reverse transcriptase, T7 RNA polymerase, a DNAendonuclease, DNA polymerase, and dNTP.